Real-time RT-PCR analysis of housekeeping genes in human skeletal muscle following acute exercise

• Published in: Physiological genomics 2004-07, Vol.18 (2), p.226-231
• Main Authors: Mahoney, Douglas J, Carey, Kate, Fu, Ming-Hua, Snow, Rodney, Cameron-Smith, David, Parise, Gianni, Tarnopolsky, Mark A
• Format: Article
• Language: English
• Subjects: Adult; Computer Systems; Exercise - physiology; Gene Expression Regulation - physiology; Genes - physiology; Humans; Male; Muscle, Skeletal - physiology; Quality Control; Reverse Transcriptase Polymerase Chain Reaction - methods
• ISSN: 1094-8341; 1531-2267
• DOI: 10.1152/physiolgenomics.00067.2004

1 Department of Medical Sciences, McMaster University, Hamilton, Ontario, Canada L8N 3Z5 3 Department of Kinesiology, McMaster University, Hamilton, Ontario, Canada L8N 3Z5 4 Department of Pediatrics, McMaster University, Hamilton, Ontario, Canada L8N 3Z5 2 School of Health Sciences, Deakin University, Burwood, Victoria 3125, Australia Studies examining gene expression with RT-PCR typically normalize their mRNA data to a constitutively expressed housekeeping gene. The validity of a particular housekeeping gene must be determined for each experimental intervention. We examined the expression of various housekeeping genes following an acute bout of endurance (END) or resistance (RES) exercise. Twenty-four healthy subjects performed either a interval-type cycle ergometry workout to exhaustion ( 75 min; END) or 300 single-leg eccentric contractions (RES). Muscle biopsies were taken before exercise and 3 h and 48 h following exercise. Real-time RT-PCR was performed on ß-actin, cyclophilin (CYC), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and ß2-microglobulin (ß2M). In a second study, 10 healthy subjects performed 90 min of cycle ergometry at 65% of O 2 max , and we examined a fifth housekeeping gene, 28S rRNA, and reexamined ß2M, from muscle biopsy samples taken immediately postexercise. We showed that CYC increased 48 h following both END and RES exercise (3- and 5-fold, respectively; P < 0.01), and 28S rRNA increased immediately following END exercise (2-fold; P = 0.02). ß-Actin trended toward an increase following END exercise (1.85-fold collapsed across time; P = 0.13), and GAPDH trended toward a small yet robust increase at 3 h following RES exercise (1.4-fold; P = 0.067). In contrast, ß2M was not altered at any time point postexercise. We conclude that ß2M and ß-actin are the most stably expressed housekeeping genes in skeletal muscle following RES exercise, whereas ß2M and GAPDH are the most stably expressed following END exercise. gene expression; endogenous controls; validation